! This Administrative Supplement (AS) in response to PA-18-591 is designed to test the hypothesis that carbonic anhydrase-8 (CA8) overexpression using HSV CA8 viral gene therapy (vHCA8) may represent a novel therapy for AD. vHCA8 biotherapeutic development is currently supported by non-AD NINDS NIH IGNITE Award R21NS105880 funding. CA8 is an allosteric of inhibitor of neuronal inositol trisphosphate receptor-1 (ITPR1) and ryanodine receptor (RYR). Calcium dysregulation is believed to be one of the molecular lynchpins of AD pathogenesis. Accumulation of A?-aggregates at diseased neurons is believed to be responsible for this calcium dysregulation and global sustained increases of intracellular calcium dependent on ITPR and RYR calcium release channels. CA8 lowers free calcium through inhibition of IPTR1- and RYR- mediated calcium release that is fundamental to neuronal excitability, synaptic functioning, genomic programming, neuronal degeneration and ultimately dementia. Our current HSV biotherapeutic is well adapted for long-term expression of reporter genes in all neurons including those in the CNS, making this technology potentially highly relevant to AD. In Aim 1 we will test the hypothesis that vHCA8 replication defective viral constructs inhibit A?1-40 & 1-42 oligomers induced calcium-signaling dysregulation in vitro. We will utilize 3 novel vHCA8 vectors from our NINDS Award including: (1) CMV promoter driving CA8WT expression (positive control); (2) CMV promoter driving CA8MT expression (no CA8 protein); and (3) CMV promoter driving mCherry reporter without CA8 (2 & 3 are negative controls) controlling for A?-induced neuronal degeneration (toxicity). We will assess for ITPR1 and/or RYR activation (pITPR1, pRYR), tauopathy (pTau) and calcium dysregulation; and whether vCA8 can inhibit these. In Aim 2 we will test the hypothesis that vHCA8 gene therapy will inhibit mutant A? precursor protein (A?PPM) and PSEN1 (PSEN1 ?E9)-induced A? and tau pathology in 3D culture. The same three novel vHCA8 viral constructs will be used to determine if A? aggregates and tau tangle overproduction due to lentiviral A?PPM and PSEN1 ?E9 overexpression can be inhibited by vHCA8 using westerns and immunohistochemistry (pITPR1, pRYR, pTau, A?-aggregates). Our vHCA8 biotherapeutic candidates embody a novel approach to prevent or inhibit AD pathogenesis by correcting calcium dysregulation associated with AD. If these novel pilot studies are successful, they will have a major impact on our understanding of AD pathogenesis and provide an early proof-of-concept for CA8 biotherapeutic applications with the potential to transform the care of AD patients. These data may lead to more comprehensive scientific AD proposals, intellectual property creation, and the testing of novel therapeutic approaches to prevent or treat AD.